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Biomedicum Genomics Oy
support@bmgen.fi
Haartmaninkatu 8
FI-00290 Helsinki
Finland
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Quality Control of Recombinant Virus Products
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The quality and biosafety of the produced recombinant viruses can be
assessed by the following tests:
Exclusion of replication competent viruses (RCV test)
Capsid titer test (p24)
Live virus titer analysis (IU/ml)
Exclusion of replication competent viruses (RCV test)
Recombinant retro- and lentiviruses are designed to perform only one round
of infection. After the infection, a segment of the transfer vector that
encodes shRNA/miRNA or cDNA integrates into the host cell's genome and is
expressed under the control of the viral promoter. The recombinant viruses
do not carry genetic information for production of virus particles and
therefore, cannot replicate or produce new virus particles in the target
cells. However, there is a very small likelihood that unusual
recombination events lead to formation of replication competent viruses
during the virus production. We exclude the presence of replication
competent virus (RCV test) in lentivirus preparations upon request. RCV
test can also be performed to transduced cells to ensure that the cells
are virus-free before transferring cells outside from the Biosafety Level
2 laboratory. BMGen provides individual RCV tests as a service.
Capsid titer test (p24)
A 24 kilodalton protein (p24) is a major structural core component in the
recombinant lentiviruses. We use a p24 specific enzyme-linked
immunosorbent assay (ELISA) to measure the p24 lentivirus capsid protein
concentration (pg/ml) from the cell culture supernatants containing
lentivirus preparations. Together with live virus titer analysis (IU/ml)
this can be used to assess the quality of the products.
Live virus titer analysis (IU/ml)
The virus constructs containing fluorescent protein coding sequence, such
as GFP, can be used to analyze the amount of infectious units (IU) in the
sample and give an approximation of the transduction efficiency of the
virus particles. By calculating the percentage of fluorescence-positive
(and thus infected) cells and comparing this to the used virus supernatant
volumes, dilutions and cell amounts, the infection efficiency of the virus
particle batch can be measured. As an example, a test value of 1 x 10^5
IU/ml means that 1 ml of virus particles can infect 100.000 cells or that
10 µl of virus particles is sufficient to infect 1000 cells. The live
virus titer values are specific to cell types, and different cell types
vary in their susceptibility to infection. We use HeLa cells to measure
the IU/ml values.
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